In striated muscle, the thin filament consists of polymerized actin, which is decorated with the regulatory proteins troponin C (TnC) and tropomyosin. TnC is a single-polypeptide protein with a molecular weight near 18,000. We study the regulatory domain of TnC from chiken skeletal muscle using genetically generated mutants which contain a single tryptophan at positions 22, 52, and 90. The quantum yields of Trp-22 are 0.33 and 0.25 in the presence of Mg2+ (2-Mg state) and Ca2+ (4-Ca state), respectively. The anisotropy decay of of all three tryptophans show two rotational correlation times. The long correlation time observed for Trp-22 and Trp-90 suggest an asymmetric hydrodynamic shape. Data obtained at the CFS showed that TnC becomes more asymmetric upon binding the activator Ca2+.